Fluorescence microscope

ABSTRACT

Fluorescence microscope with blocking filters for a portion of the light emitted by a specimen have markings referring to the orientation of their wedge angle. A marking to which the marking on the filter can be oriented in a defined manner is preferably provided on the filter holder of the microscope, the filters being marked with respect to their wedge angle.

[0001]FIG. 1 shows the beam path in a microscope equipped forfluorescence applications. The light from an additional light source (1)passes through a heat-absorbing filter (2), red attenuating filter/stopslide (3) and a field diaphragm (4) to the excitation filter (5). Thelatter is installed in the reflector slide of the microscope which alsocontains a dichroic beam splitter (6). The dichroic beam splitterreflects the shortwave excitation light through the objective (7) intothe specimen or preparation (8).

[0002] The occurring emission is collected by the objective (7)and—because it has greater wavelengths than the excitation lightis—passed by the dichroic beam splitter (6). The beams now pass throughthe emission filter (9). The remainder of the excitation light isfiltered out by the latter. For this reason, this filter is alsoreferred to as a blocking filter. As is conventional, the tube lens (10)and eyepiece (11) form the microscope image formed of fluorescent light.

[0003] In order to avoid image offset (pixel shift), multiple exposuresin fluorescence recordings with different emission filter sets (A, B)require an optimal congruence of the object image in the individualrecordings. However, there are technological limits in this respect.

[0004] Because of the different wedge angles of the emission filters(A_(EM), B_(EM)) and of the color splitters, the filter combinationsneeded for the fluorescence application cause a slight image offset.This is shown in FIG. 2.

[0005] The reference symbols have the following meanings:

[0006] A_(Em) emission filter of filter set A

[0007] B_(Em) emission filter of filter set B

[0008] a₁ light beam striking A_(Em)

[0009] b₁ light beam striking B_(Em)

[0010] a₂ light beam deflected by A_(Em)

[0011] b₂ light beam deflected by B_(Em)

[0012] α_(A) angle between the incident light beam a₁ and the deflectedlight beam a_(c) of filter A_(EM)

[0013] α_(B) angle between the incident light beam b₁ and the deflectedlight beam b₂ of filter B_(EM)

[0014] E image plane

[0015] {overscore (P_(A)P_(B))} distance (pixel shift) between the imagepoints impinging on the image plane E

[0016] The light beams a₁ and b₁ impinge on the emission filters A_(Em)and B_(Em) of the corresponding filter sets A and B. The beam isdeflected in more or less opposite directions because of the existingwedge angle of the filters depending on the installed position (a₂ andb₂ are greatly exaggerated in the drawing in order to illustrate theprocess). Therefore, the image points impinging on the image plane E donot lie exactly one above the other, but are offset relative to oneanother by the pixel shift. Even with the close tolerances of thefilters sets by Carl Zeiss with a slight image offset, this offset stilloccurs to a slight extent.

[0017] According to the invention, as is shown in FIG. 3, the filtersare aligned with one another with respect to their wedge angle. Thefilters are measured and marked by the microscope manufacturerbeforehand with respect to wedge angle and orientation, for example, inan autocollimator, e.g., by means of a line S on the side which can bearranged, e.g., on the side located opposite the deflecting directionthrough the wedge effect. When the filter is inserted into therespective filter module of the microscope, this filter module also hasa marking which is made to coincide with the marking on the filter.Identical orientation of the filters is ensured in this way.

[0018] After the emission filters A_(Em) and B_(Em) are swiveled in (seeFIG. 1), the impinging light beams a₁ and b₁ are deflected in the samedirection (a₂ and b₂). In this way, the pixel shift which exists to aslight extent in any case is minimized or, ideally, compensated (pixelshift {overscore (P_(A)P_(B))}′).

[0019] In this connection, the wedge angles can also be determined onthe part of the manufacturer and filters with identical wedge angles canbe marked and correlated by the user.

1. Fluorescence microscope with blocking filters for a portion of thelight emitted by a specimen which are marked with respect to theorientation of their wedge angle.
 2. Fluorescence microscope accordingto claim 1, wherein a marking to which the marking on the filter can beoriented in a defined manner is provided on the filter holder of themicroscope.
 3. Fluorescence microscope according to claim 1 and/or 2,wherein the filters are marked with respect to their wedge angle.